Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.
A class of compounds suitable for this task is insulin derivatives in which the ε-amino group in the lysine residue in position 29 of the B-chain is acylated with a hydrophobic moiety. Such compounds are disclosed in EP patents 792,290 and 894,095 and in U.S. Pat. Nos. 5,693,609, 5,646,242, 5,922,675, 5,750,497 and 6,011,007.
Human insulin and closely related insulins have three primary amino groups in the molecule namely the α-amino groups of GlyA1 and PheB1, respectively, and the ε-amino group of LysB29. N-Acylation of unprotected insulin may—depending on the conditions—lead to a complex mixture of mono-, di- and even triacylated products. However, although a certain preference for acylation of a specific position can often be observed the preference is not always sufficiently pronounced to make such direct acylation useful as a method of producing monoacylated insulins since the formation of the desired monoacylated product may be accompanied by the formation of considerable amounts of closely related unwanted by-products such as di- and tri-acylated insulins. When such unwanted are formed, this happens at the expense of the desired product and may lead to problems in the purification of the desired product.
Acylation of only one or two specific amino groups in the insulin molecule can be achieved if a suitably protected intermediate is available. A suitable intermediate can be an insulin molecule in which the amino group(s) not to be acylated is (are) blocked with a protection group which can be removed selectively after the acylation has been performed. Such a protected intermediate can be an insulin molecule in which one or two protection groups have been introduced posttranslationally in a specific way. For economic reasons, it is however very attractive to avoid the use of specific protection groups if possible.
Selective acylation at pH above 9 by use of an activated ester of a fatty acid of, in particular, a free ε-amino group in either position B29 or in position B28 is disclosed on U.S. Pat. No. RE 37971. U.S. Pat. No. 5,905,140 discloses selective acylation of a free ε-amino group in insulin by used of an activated amide.
The present invention is related to a process for obtaining high yields of insulin or an insulin analogue being acylated in an ε-amino group, in particular the ε-amino group in LysB29.